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|標題:||XpsE N端區域位於 XpsE-XpsE相互作用介面的分析
The N-terminal region of XpsE is located at the interface of protein-protein interaction with itself
|關鍵字:||The N-terminal region of XpsE;XpsE N端區域位||出版社:||生物化學研究所||引用:||Abendroth J, Murphy P, Sandkvist M, Bagdasarian M, Hol WG (2005) The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J Mol Biol 348: 845-55 Bleves S, Voulhoux R, Michel G, Lazdunski A, Tommassen J, Filloux A (1998) The secretion apparatus of Pseudomonas aeruginosa : identification of a fifth pseudopilin, XcpX (GspK family). J Biol Chem 271: 2701-2708. Chen LY, Chen DY, Miaw J, Hu NT (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem 271: 2703-8 Chien IL (2002) Purification and characterization of the XpsE protein of the type II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Chen Y, Shiue SJ, Huang CW, Chang JL, Chien YL, Hu NT, Chan NL (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem 280: 42356-63 Camberg JL, Sandkvist M (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187: 249-56 Dums F, Dow JM, Daniels MJ (1991) Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet. 229: 357-364. Douville K, Price A, Eichler J, Economou A, Wickner W (1995) SecYEG and SecA are the stoichiometric components of preprotein translocase. J Biol Chem 270: 20106-20111. Filloux A (2004) The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694: 163-179 Genin S, Boucher, CA (1994) A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mol Gen Genet 243: 112-118 Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, Wu CY (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol 174: 2679-2687 Hu NT, Hung MN, Liao CT, Lin MH (1995) Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology 141: 1395-406 Hu NT, Leu WM, Lee MS, Chen A, Chen SC, Song YL, Chen LY (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J 365: 205-211 Lee HM, Tyan SW, Leu WM, Chen LY, Chen DC, Hu NT (2001) Involvement of the XpsN protein in formation of the XpsL-XpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol 183: 528-535 Lee MS, Chen LY, Leu WM, Shiau RJ, Hu NT (2005) Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. J Biol Chem 280: 4585-4591 Nunn DN, Lory S (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol 75: 4375-82 Pugsley AP (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57: 50-108. Py B, Loiseau L, Barras F (1999) Assembly of the type II secretion machinery of Erwinia chrysanthemi: Direct interaction and associated conformational change between OutE, the putative ATP binding component and the membrane protein OutL. J Mol Biol 289: 659-670 Pang (2008) The biochemical analysis of coexpressed and copurified XpeE/MBP-XpsLN complex. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Sandkvist M, Bagdasarian M, Howard SP, DiRita VJ (1995) Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J 14: 1664-1673 Sandkvist M, Keith JM, Bagdasarian M, Howard SP (2000) Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae. J Bacteriol 182: 742-8 Sandkvist M (2001) Biology of type II secretion. Mol Microbiol 40: 271-83 Shiue SJ, Kao KM, Leu WM, Chen LY, Chan NL, Hu NT (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25: 1426-35 Satyshur KA, Worzalla GA, Meyer LS, Heiniger EK, Aukema KG, Misic AM, Forest KT (2007) Crystal structures of the Pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Cell 15: 363-376 Thomas JD, Reeves PJ, Salmond GP (1997) The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF. Microbiology 143: 713-20 Tsai RT, Leu WM, Chen LY, Hu NT (2002) A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J 367: 865-71 Tseng YN (2006) Significance of the N-terminal domain of XpsE in its interaction with XpsL analyzed by site-directed mutation. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Wei CJ (2006) Analysis of conformation change at the N domain of XpsE protein by utilizing fluorescence probes. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Yo TT (2003) Detection of interactions between XpsF and XpsL, or XpsE, in the type II secretion apparatus of Xanthomonas campestris pv. Campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. Yamagata A, Tainer JA (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J 26: 878-890 Watanabe Y, Takano M, Yoshida M (2005) ATP binding to nucleotide binding domain (NBD)1 of the ClpB chaperone induces motion of the long coiled-coil, sbilizes the hexamer, and activates NBD2. J Biol Chem 280: 24562-24567||摘要:||
十字花科黑腐病菌的第二型分泌機器由12個蛋白組成，其中 XpsE 是唯一位在細胞質內的非膜蛋白，具有微弱的 ATPase 活性；XpsE 可區分為 N 端及 C 端兩個獨立區域，先前研究指出，XpsE 的 N 端是與 XpsL 結合的決定性因子。已解出的 XpsE N 結晶結構有兩種: 兩者之間因位於 N 端的 α1、α2 兩個 helices 與 α3 相對位置不同而被命名為 “open” 及 “closed” form；closed form 的特徵是具有一個open form 中所沒有的hydrophobic patch，此 hydrophobic patch 曾被推測為可能參與 XpsE 與 XpsL 之間的交互作用。本研究藉由 thiol-specific hetero-bifunctional crosslinkers 來分析 XpsE 蛋白與其相互作用蛋白間交互作用的介面。功能分析顯示針對位於 hydrophobic patch 的三個疏水性胺基酸進行定點突變後 XpsE(Cys-, V11C)、XpsE(Cys-, L25C)、XpsE(Cys-, L39C) 都仍維持 XpsE 的正常功能。單獨表現 XpsE(Cys-, V11C)、XpsE(Cys-, L25C)、XpsE(Cys-, L39C) 突變蛋白或各別與 MBP-XpsLN 共表現的蛋白複合體，與 crosslinkers SIA 或 SPDP 作用後，均能在 Coomassie blue 染色膠圖中觀察到分子大小約為 crosslinked dimer 的訊號；經由 Western blot 顯示此 crosslinked dimer 可能主要源自 XpsE-XpsE 間的交互作用形成。對照組實驗顯示 XpsE(Cys-, V11C)、XpsE(Cys-, L25C)、XpsE(Cys-, L39C) 在不加 crosslinker 時也形成對還原劑敏感的 dimer，推測這的訊號是由 disulfide bond 鍵結而成，且可能分別由相鄰 XpsE 分子中的 V11C、L25C、L39C 相互鍵結而成。進一步利用氧化劑 Cu-phenanthroline 處理後，發現氧化劑皆能促進此 disulfide bond dimer 的訊號。透過 size exclusion chromatography 觀察蛋白分子大小的分佈時，發現 XpsE(Cys-, V11C)、XpsE(Cys-, L39C) 突變蛋白六聚體的比例比XpsE(Cys-) 較多，XpsE(Cys-, L25C) 則否。暗示這樣 disulfide bond 鍵結成的 dimer 可穩定 XpsE 蛋白形成的六聚體結構。綜合以上結果推論，位於 XpsE N 端的 hydrophobic patch 中之胺基酸 V11 及 L39 位於相鄰 XpsE 的交互作用介面。
The Xanthomonas campestris pv. campestris type II secretion system comprises of 12 protein components. Of all components, XpsE is the only cytoplasmic protein with weak ATPase activity. The N domain of XpsE (XpsEN) acts as the determinant in its association with XpsL. Two crystal structures of XpsEN have been resolved, an open and a closed form. They differ in the position of α1、α2 relative to α3 at their N-termini. A hydrophobic patch surrounded by residues 11, 25 and 39 was detected only in the closed form. It has been suggested to be involved in the interaction between XpsE and XpsL.. In this study I used two different thiol-specific hetero-bifunctional crosslinkers to examine if the three residues surrounding the hydrophobic patch interact directly with XpsL. Mutation of each residue into cysteine in a cys-less XpsE did not cause any effect in the normal function of XpsE for secretion. Each mutated XpsE, when expressed alone or co-expressed with MBP-XpsLN for isolating XpsE/MBP-XpsLN complex, was treated with the crosslinker SIA or SPDP and analyzed in SDS-polyacrylamide gel. Signals with apparent molecular weight of crosslinked dimer were detected in each case. Detailed analysis indicated that the crosslinked dimer originated from XpsE-XpsE crosslinking, not from XpsE-XpsLN crosslinking. Dimer-sized signal that was sensitive to reducing agent was detected in the control sample, into which no crosslinker was added. These observations implied disulfide bond formation between two cysteines located in neighboring XpsE. The proposition was confirmed by significant increase in the dimer signal when each mutated XpsE was incubated with the oxidizing agent Cu-phenanthroline. Analysis of size distribution of monomeric and oligomeric XpsE through size exclusion chromatography revealed that two mutated proteins XpsE(Cys-, V11C) and XpsE(Cys-, L39C), but not XpsE(Cys-, L25C), exhibited elution profiles with oligomer in higher proportion than that of the cys-less XpsE. It appears that XpsE hexamer was stabilized by L39C- or V11C-mediated dimer formation. In summary, the experimental evidences presented in this study suggested at least two residues surrounding the N-terminal hydrophobic patch observed in the closed form crystal structure of XpsEN, V11 and L39, are located at the interactive interface of two neighboring XpsE.
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